Novel and Bioactive Natural Products from the Marine-Derived Endophytic Fungi

Abstract

The main goal of this study was to discover novel natural products with pharmacological potential by employing various biotechnological methods. Marine fungi have been identified as a promising resource for such molecules. In particular, fungi living in the inner tissue of marine algae and sponges (endophytes) were found capable of producing a structurally and with regard to their bioactivity, most intriguing array of compounds.<br /> This study thus focussed on the three marine-derived fungi Coniothyrium cereale, Phaeosphaeria spartinae and Auxarthron reticulatum. They were cultivated in artificial media mimicking the marine environment and investigated for their bioactive secondary metabolites. The investigation dealt with the isolation, identification and biological evaluation of the fungal natural products with emphasis on compounds exhibiting cytotoxic and antimicrobial activities. Additionally, natural products that inhibit proteases such as Human Leukocyte Elastase (HLE), and compounds with affinity toward cannabinoid receptors were explored.<br /> The extracts of C. cereale, P. spartinae and A. reticulatum were subjected to purification employing high-tech chromatographic techniques. The isolated metabolites were characterized based on the extensive spectroscopic measurements. Stereochemical assignments were done through X-ray crystallographic and CD spectral analysis. The identified compounds were then evaluated for their biological activities. Over all 36 interesting compounds, partly with unprecedented chemical structures and outstanding bioactivity were isolated from three different fungal strains.<br /> The detailed analysis of the marine endophytic fungus C. cereale resulted in a series of compounds with a polyketide skeleton, i.e. the phenalenones. From the structural point of view, the most interesting compounds are the nitrogenous metabolites, i.e. conio-azasirosterol, cereo-azasirosterol, cereolactam and cereoaldomine. Conio-azasirosterol and cereo-azasirosterol are most unusual heterodimers composed of two different skeletons of natural products. In compounds cereolactam and cereoaldomine the nitrogen is either forming a rare γ-lactam ring or imine group, respectively. The Conitothyrium metabolite cereoazulene is a unique dioxa-azulene derivative, probably of polyketide origin and formed through oxidative cleavage of a regular polyketide precursor molecule. The detailed analysis of the marine endophytic fungus Ph. spartinae resulted in a novel steroidal compound and a series of polyketides. Spartogesterone is an unusual steroid due to the presence of a carboxylic group at C-4 and the structural similarity to the hormone progesterone. Indeed, the human hormone progesterone has only recently (2010) been discovered in plants, and the finding in our study suggests that progesterone derivatives also do occur in fungi. The polyketides spartinols A-D are characterized by the presence of a central cyclohexene ring to which two side chains are attached, long one with a trans triene system and another short one, substituted with hydroxyl-, carbonyl- and /or carboxyl group. This side chain also can be cyclized to form a furanoid ring, as seen in furanospartinol and a pyranoid ring as present in pyranospartinol. Pyranospartinol is also different from the aforementioned metabolites by having a cis diene double bond instead of a trans triene. Spartinoxide which was also obtained from this fungus has an uncommon allene moiety and epoxy group.<br /> From the fungus A. reticulatum two promising alkaloids were obtained, i.e. amauromine and methyl-penicinoline. Amauromine is an alkaloid with a C-2 axis of symmetry. It is composed of two prenylated tryptophan moieties, which are condensed to form a diketopiperazine nucleus. Methyl-penicinoline is a structurally unique 4-quinolinone, linked to a pyrrole ring on one side and to a methyl carboxylic acid ester moiety on the other.<br /> In antimicrobial assays, compounds conioscleroderolide, coniosclerodione, (–)-cereolactone, and (–)-scleroderolide showed activity against Staphylococcus aureus SG 511 with MIC values of 23.8, 65.7, 52.0, and 23.8 μM, respectively. In agar diffusion assays with Mycobacterium phlei considerable inhibition zones (> 15 mm) were observed for Z-coniosclerodinol, (S,S)-sclerodinol and coniolactone. (–)-Trypethelon strongly inhibited the growth of M. Phlei, S. Aureus and E. coli with inhibition zones of 18, 14 and 12 mm, respectively. <br /> In cytotoxic assays, using an MTT assay with mouse fibroblast cells, the compounds (–)-sclerodione and (–)-trypethelone had significant activity with an IC50 value of 6.4 and 7.5 µM, respectively. Cytotoxicity was also determined using an epithelial bladder carcinoma cell line, in which the compounds conioscleroderolide and (–)-scleroderolide exhibited very weak in vitro cytotoxicity with IC50 values of 27 and 41 μM, respectively. Coniosclerodin, conioscleroderolide, (–)-sclerodin, (–)-cereolactam, (–)-cereoaldomine, spartinoxide and prenyl-hydroxyl-benzoic acid showed potent inhibition of the protease HLE with IC50 values of 7.16, 13.3, 10.9, 9.28, 3.01, 6.5 and 8.1 μM, respectively. <br /> Amauromine displayed a potent and selective antagonistic activity toward CB1 receptors with a Ki value of 178 nM. No affinity was noted for CB2 receptors. To the best of our knowledge, amauromine is the first compound of fungal origin to have affinity to cannabinoid receptors, and it is the first exogenous peptide to have such an activity.<br /> In conclusion, the chemical investigation of marine fungi living in algal and sponge tissues resulted in the discovery of structurally novel natural products with interesting biological activities. In fact this study contributed novel structural skeletons probably useful as lead drugs for the development of cytotoxic antibiotic compounds, protease inhibitors and cannabinoid receptor antagonists.